Green‑Synthesized Silver Nanoparticles from Pomegranate Peel: Potent Antimicrobial and Colon‑Cancer Cytotoxicity

Background

Nanotechnology’s rapid evolution has spotlighted green synthesis—using plant extracts—to produce nanoparticles below 100 nm, ideal for drug delivery and biomedical applications. Unlike costly chemical or physical routes, the green approach is inexpensive, non‑toxic, and harnesses natural reducing agents such as proteins, enzymes, and carbohydrates. Silver nanoparticles (AgNPs) produced this way display superior antimicrobial efficacy, attributable to their high surface‑area‑to‑volume ratio and ability to penetrate bacterial cells more efficiently than silver ions.

Hospital‑acquired infections, often driven by Gram‑negative and Gram‑positive bacteria, necessitate new antibacterial strategies. Recent studies show that AgNPs can synergize with conventional antibiotics, enhancing overall activity. Moreover, AgNPs have demonstrated cytotoxic effects on various cancer cell lines, making them attractive candidates for anticancer therapy.

This research focuses on synthesizing AgNPs from Punica granatum peel extract, evaluating their antibacterial potency against a panel of clinically relevant pathogens, and assessing cytotoxicity on a colon cancer cell line.

Methods

Preparation of the Peel Extract

One kilogram of Saudi pomegranate fruits (cultivated in Taif) was washed, peeled, and dried. The peel was ground into fine powder, and 10 g was soaked in 100 mL double‑distilled water for 24 h at room temperature. The mixture was filtered through Whatman No. 1 paper to obtain the aqueous extract, performed under sterile conditions.

Synthesis of AgNPs

0.1 mM silver nitrate was mixed with 250 mL double‑distilled water. Ten milliliters of the peel extract was added, and the solution was shaken for 5 min. A color change from colorless to dark brown, observed after 24 h, indicated Ag⁰ formation. The suspension was centrifuged at 15,000 RPM for 15 min, repeated four times, yielding purified AgNPs for subsequent analyses.

Characterization

UV‑Vis spectra (200–800 nm) were recorded at 24, 48, and 72 h using a Perkin Elmer Lambda 950. X‑ray diffraction (PANalytical) determined crystal structure. TEM (JEOL JEM‑1230) and SEM (JSM 6380 LA) assessed morphology and size; EDX (JED 2200) quantified elemental composition.

Antibacterial Assays

Bacterial Suspensions

Clinical isolates (E. coli ATCC 25922, P. aeruginosa ATCC 27584, P. vulgaris ATCC 8427, S. typhi ATCC 14028, S. aureus ATCC 29213, S. epidermidis MTCC 3615, K. pneumoniae) were revived, cultured on nutrient agar, and prepared at 10⁶ CFU mL^‑1.

Agar Well Diffusion

Wells were filled with 25, 50, 75, and 100 µL of AgNP suspension. Plates were incubated at 37 °C for 24 h, and inhibition zones (mm) were measured. Each assay was performed in triplicate.

Cytotoxicity

Cell Proliferation

RKO colon cancer cells (ATCC CRL‑2577) were seeded at 5 × 10³ cells mL^‑1 in 96‑well plates and treated with 0.3–100 µg mL^‑1 AgNPs. Alamar Blue assays were conducted on days 3 and 5, measuring relative fluorescence units.

Apoptosis/Necrosis

Cells were stained with acridine orange/ethidium bromide (AO/EtBr) after 5 days of exposure to 1.5–25 µg mL^‑1 AgNPs and examined under a fluorescence microscope to identify viable, apoptotic, and necrotic cells.

Statistical Analysis

Data were analyzed using one‑way ANOVA (GraphPad Prism 6.0). Results with p < 0.05 were considered statistically significant.

Results and Discussion

The green synthesis proceeded smoothly, with a visible color shift confirming AgNP formation. UV‑Vis spectra displayed a characteristic plasmon peak at 378 nm, whose intensity increased over time, reflecting higher nanoparticle concentrations.

XRD patterns matched the face‑centered cubic structure of silver (JCPDS No. 04‑0783). TEM images revealed uniformly distributed, spherical particles ranging from 20 to 40 nm, averaging 26.95 nm. SEM confirmed similar morphology, while EDX analysis quantified 70% Ag by weight, confirming successful synthesis.

Antibacterial testing showed dose‑dependent inhibition zones. Gram‑negative bacteria (E. coli, P. aeruginosa, S. typhi) and Gram‑positive bacteria (S. aureus, S. epidermidis, K. pneumoniae) were inhibited, with the most pronounced effects observed at 100 µL concentrations. Notably, S. typhi exhibited lower susceptibility, aligning with previous reports.

Cytotoxicity assays revealed a 56% reduction in RKO cell viability on day 3 and 61% on day 5 with 12.5 µg mL^‑1 AgNPs. AO/EtBr staining confirmed apoptosis and autophagy‑related changes, indicating that these nanoparticles can trigger programmed cell death pathways in colon cancer cells.

Conclusions

Pomegranate peel extract serves as an effective, sustainable reducing agent for producing AgNPs with optimal size (20–40 nm) for drug delivery. The synthesized particles exhibit strong antibacterial activity across a spectrum of clinically relevant pathogens at low concentrations and demonstrate significant cytotoxicity against colon cancer cells. These findings position green‑synthesized AgNPs as promising candidates for dual antibacterial and anticancer therapeutic development.

Abbreviations

EDX

Energy‑dispersive X‑ray spectroscopy

SEM

Scanning electron microscope

TEM

Transmission electron microscopy

XRD

X‑ray diffraction